We continue to develop our biophysical measurement systems (biological atomic force microscopy (Bio-AFM) platforms, Quartz Crystal Microbalance-Dissipation (QCM-D), and optical microscopy and spectroscopy), and to apply these technologies to a number of biomedical and biological investigations in collaboration with NIH intramural and extramural scientists. On the instrumentation front, we are continuing an integration of Raman spectroscopy with Bio-AFM, and working toward higher speed and broader applicability of AFM imaging and single molecule force spectroscopy (SMFS) for cellular and biomedical sample characterizations. On the application front, our major collaborations and notable results in this year include: (A) We have maintained our commitment to work on clinical vaccine development toward enhanced immunological response and eventual eradication of malaria. We have investigated the macromolecular structure and nanomechanical properties of more malaria vaccine candidates and carriers via Bio-AFM and related bioanalysis with Dr. David Narum (Laboratory of Malaria Immunology and Vaccinology, NIAID, NIH), and other collaborators. These malaria protein antigens and vaccine carriers are being produced via recombinant-protein biotechnology, purified, and characterized in a manner suitable for human trials and scale-up production. Biophysical characterization at single macromolecular level using AFM imaging and force spectroscopy are helping define these biologics through their developmental phases. We also aim to improve mechanistic understanding of the malaria parasites and pathogen-host interactions. (B) We have expanded our collaboration on multifunctional nanomedicine probes with Dr. Xiaoyuan Chen (laboratory of Molecular Imaging and Nanomedicine, NIBIB), Dr. Ashwin Bhirde, Dr. Dingbin Liu, and an international team of co-investigators. New results on graphene oxide based theranostics and gold nanoparticle based imaging and biomarker detection probes are among those published this year. Other results on theranostics based on carbon nanotubes, nanodots and other nano-constructs have been developed further toward multiple cellular imaging and biomedical applications. We are examining broader applications of Bio-AFM and QCM-D methodology for investigating nanoparticle theranostics and their impact on cancer cells, stem cells, and related biomedical systems. (C) We have continued our Bio-AFM studies of protein clathrin and assemblies with collaborators including Dr. Ralph Nossal (NICHD, NIH) and Prof. Eileen Lafer (Univ. Texas Health Sciences Center, San Antonio). Clathrin is a key protein for receptor-mediated endocytosis and intracellular trafficking and we reported on clathrin-clathrin interactions and intra-clathrin domain folding energetics using single molecule force spectroscopy (SMFS) during this year. Further AFM and QCM-D measurements are been pursued to characterize clathrin and its assembled structures toward understanding its function from yeast to human. (D) We have collaborated with NIDCR scientists, Dr. Andrew Doyle, Ms. Jill Harunaga and Dr. Kenneth Yamada (Laboratory of Cell and Developmental Biology, NIDCR) on nanomechanics and structural properties of tissue specific extracellular matrices and reconstituted matrix-like gels via Bio-AFM force spectroscopy. Collaborating also with Dr. Raimon Sunyer (NICHD and currently at Institute for Bioengineering of Catalonia, Spain), we are defining the best force spectroscopy approaches to explore biological matrices and their implications in cell biology and tissue differentiation. (E) Among other collaborations this year, we have investigated protein and DNA interactions involved in nucleosome structure and dynamics, critically important in gene regulation and disease mechanisms, with Dr. Yawen Bai (Laboratory of Molecular Cell Biology, NCI) and coworkers. We have collaborated with Prof. Xiangyun Qiu (George Washington University) and his group on DNA cross-linking and related investigations. We have also advanced our Bio-AFM and biophysical studies of different protein fiber assemblies with collaborators including Dr. Richard Hendler(NHLBI), Ms. Katelyn Nagy (NCI), and Dr. Joel Schneider (NCI).